Project Summary: This program will have a major impact in the treatment of cardiovascular disease (CVD), the leading cause of death in men and women globally. Premenopausal women are less susceptible to heart disease than men, and this protection is lost after menopause. Emerging data suggest that the interplay between nitric oxide (NO) and reactive oxygen species (ROS), the nitroso-redox balance, plays a critical role in this sex-related cardioprotection. Elucidating the mechanisms underlying these gender differences is critical for development of effective therapies. S-nitrosylation (S-NO) of cysteine thiols is a major signaling pathway through which NO exerts its broad regulatory actions. It is increasingly appreciated that the underlying signaling fate of NO ultimately depends upon an intracellular state of nitroso-redox balance; such that excessive production of ROS can disrupt physiological S-NO and that CVD may reflect dysregulated protein S- NO. We have shown that the nitroso/redox balance is an important regulator of CV health and disease. Female mice have 65% more S-nitrosylated proteins in the heart6 and physiologically regulate this elevated level of S- NO with increased activity of S-nitrosoglutathione (GSNO) reductase (GSNOR), an enzyme that promotes protein denitrosylation. We previously showed that male mice lacking S-nitrosoglutathione (GSNO) reductase, an enzyme that governs S-NO levels by promoting protein denitrosylation (GSNOR??? mice) exhibit improved survival after myocardial infarction (MI). Furthermore, pregnant GSNOR?/? mice exhibit all of the clinical features of a major complication in pregnancy, preeclampsia (PE), including maternal hypertension, proteinuria and abnormal CV adaptation. Poor outcome in the GSNOR?/? females may be due to oxidative stress, as our preliminary data shows that cardiomyocytes (CMs) from GSNOR?/? females generate more ROS as compared to CMs from control females and GSNOR?/? males. Therefore, the state of increased S-NO can lead to an adverse milieu if accompanied by oxidative stress and we have uncovered a sex-associated relationship between cardiac pathology and intra-cellular nitroso-redox imbalance, which is dependent on NO and ROS production and S-NO formation and denitrosylation. Our central hypothesis is that the absence of GSNOR produces a sex-specific, antioxidant milieu in the setting of normal ROS levels (i.e., males) but one of nitrosative/oxidative stress when ROS is elevated (i.e., females). Our proposal aims to identify the fundamental mechanisms underlying this dichotomy. The specific aims are to test the hypotheses that 1) differences in nitroso-redox protein signaling determine sex-specific responses to CV stress; 2) nitroso-redox imbalance in cadiomyocytes during pregnancy underlies pregnancy-related CVD; and 3) GSNOR produces sex-specific changes in the S-nitrosylation of proteins that alter CV function. Together these aims will provide novel insights into nitroso-redox regulation of cardiac function in response to physiologic and pathophysiologic stressors and have significant implications for the treatment of CVD in men and women.